CD11b expression distinguishes sequential stages of peritoneal B-1 development

Abstract

Peritoneal cavity (PerC) B-1 cells have long been known to express CD11b, which is coexpressed with CD18 to form the Mac-1/CR3 complement receptor and adhesion molecule. However, although all PerC B-1 cells are commonly believed to express CD11b, we show here that nearly half of the cells in each of the PerC B-1 subsets (B-1a and B-1b) do not express this surface receptor. The CD11b(+) cells in each B-1 subset are larger and more granular and express higher levels of surface IgM than the CD11b(-) B-1 cells. In addition, the CD11b(+) B-1 cells initiate the formation of tightly associated doublets that are present at high frequency in adult PerC. Finally, and most importantly from a developmental standpoint, the CD11b(+) B-1 cells have a limited reconstitution capability: when sorted and transferred into congenic recipients, they reconstitute their own (CD11b(+)) B-1 subset but do not reconstitute the CD11b(-) B-1 subset. In contrast, CD11b(-) B-1 cells transferred under the same conditions efficiently replenish all components of the PerC B-1 population in appropriate proportions. During ontogeny, CD11b(-) B-1 cells appear before CD11b(+) B-1 cells. However, the clear phenotypic differences between the neonatal and adult CD11b B-1 subsets argue that although CD11b(-) B-1 give rise to CD11b(+) B-1 in both cases different forces may regulate this transition.

Fig. 1.

Successive gating scheme for identifying the CD11b⁺ and the CD11b⁻ subset on mouse B-1 cells. Total BALB/c adult PerC cells were stained according to the 11-color stain combinations described in Materials and Methods. Live cells were gated to include only lymphocytes (FSc^low, SSc^low), for which data are shown. The full gating sequence is included for on-line viewing.

Fig. 2.

CD11b⁻ cells give rise to CD11b⁺ B-1 cells in adoptive recipients. CD11b⁻ and CD11b⁺ PerC cells were sorted from BALB/c (IgM^a allotype) as described in Materials and Methods. Each sorted population was transferred into the PerC cells of BAB/25 recipients (nonirradiated). At 10–12 days after transfer, total recipient PerC cells were harvested and stained. For analysis, live cells were initially gated to include only IgM⁺ cells and donor (IgM^a) B cells were distinguished and gated by staining with a mAb that detects IgM^a (IgH-6a), whereas recipient B were identified and gated with a mAb that detects IgM^b (IgH-6b). Each B cell population was then further gated by size to include only cells within the lymphocytes scatter gates (FSc^low/SSc^low) and analyzed for the expression level of CD11b. Only B-1 cells were detected in the donor B cell population.

Fig. 3.

CD11b is expressed at higher levels on the CD11b⁺ B-1 cells than in RAG-deficient PerC recipients. CD11b⁻ or CD11b⁺ donor cells were sorted and transferred into RAG^−/− (nonirradiated) recipients. After 10–12 days, total recipient PerC cells were harvested, stained, and analyzed according to the procedures described for Fig. 2.

Fig. 4.

CD11b⁺ B-1 cells form firmly attached doublets. Total PerC cells from adult (BALB/c × CB.17)F₁ mice (IgM^a x IgM^b) were harvested and stained according to the procedures described in Materials and Methods. Because the IgM on the B-1 cells in F₁ animals is encoded either by the parental IgM^a chromosome or the parental IgM^b chromosome, cells were stained with both anti-IgM^a (FITC) and anti-IgM^b (Texas red) reagents in addition to an anti-IgM reagent (used for sorting) that detects both IgM^a and IgM^b. For confocal microscopy, CD11b⁺ B-1 cells (CD19^high, IgM^high) that fell within the FSc^high gate were sorted onto coverslips in a 24-well plate. The sorted cells were prepared and analyzed by confocal microscopy as described in Materials and Methods.

Fig. 5.

CD11b⁺ B-1 cells arise late in ontogeny but still express the typical neonatal surface phenotype, which only gradually shifts to the adult B-1 phenotype. Total PerC cells were harvested from BALB/c mice at different ages (neonates, 10 days, 30 days, and 100 days) and stained with an 11-color combination that simultaneously detects surface expression of CD11b, IgV_H11, IgM, and CD5 (see Materials and Methods). B-1 cells were identified according to the sequential gating scheme shown in Fig. 1. (Lower) IgM and CD5 expression on the B-1 subset is shown. (Upper) CD11b and V_H11 expression on the same cells is shown.